Bovine Respiratory Disease Vaccine

ABSTRACT

The present invention relates to vaccines for treating bovine respiratory disease. Such vaccines contain a combination of bovine influenza D virus and Mannheimia haemolytica antigens. An upper respiratory infection with an IDV leads to an increased potential for M. haemolytica pathology in the lungs. The vaccines may contain further antigens from other bovine respiratory pathogens.

The present invention relates to vaccines to treat bovine respiratory disease. Such vaccines contain a combination of bovine influenza D virus antigens and Mannheimia haemolytica antigens.

Bovine respiratory disease (BRD) is the most significant disease affecting the United States cattle industry, causing annual losses estimated to be in excess of one billion dollars. Numerous commercial vaccines variably consisting of combinations of inactivated and modified live bacteria and viruses are widely used; however, the incidence of BRD has been increasing for the past several decades. Animal stress, often due to movement and animal management, is thought to predispose cattle to BRD. A number of viruses have established roles in BRD pathogenesis, including bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus, bovine parainfluenza virus 3, bovine rhinitis virus, and possibly influenza D virus (IDV) (Ferguson et al, J. Virol. 90:5636-42, 2016).

First isolated from pigs with acute respiratory disease, the proposed influenza D virus has subsequently been identified in bovines in numerous countries. Calves inoculated with IDV displayed only mild respiratory disease compared to controls, and virus replication was detected in the respiratory tract associated with a significant increase in neutrophils in the trachea. The highest viral titers were present in the nasal turbinates, decreasing in tissues lower in the respiratory tract. IDV transmitted to contact animals and all inoculated and exposed animals seroconverted. While serological surveys of swine and human sera found low antibody prevalence (9.5 and 1.3%, respectively), several studies have found that IDV antibodies are nearly ubiquitous in bovines, with up to 94% of neonatal beef cattle being seropositive for IDV, likely due to the presence of maternal antibodies. As maternal antibodies are known to interfere with vaccination and the development of active immunity, the presence of antibodies at the time of vaccination may lessen the immune response to a vaccine.

While not conclusively established, IDV may be part of the bovine respiratory disease complex (BRDC). Commercial vaccines for IDV have been suggested, for example in U.S. Pat. No. 9,278,999 and WO2014/015091, but have not been adopted, likely in part due to IDV's recent discovery and its unknown (and possibly minor) role in BRD pathogenesis. Inclusion of IDV into commercial BRD vaccines may improve their efficacy, if IDV infection exacerbates disease caused by other bovine respiratory pathogens, rather than IDV being an opportunistic infection in BRD caused by other pathogens.

The present invention provides for an immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen. The combination vaccine provides increased efficacy against BRD compared to monovalent vaccines alone. The Mannheimia haemolytica antigen may be an inactivated bacterium, or extracts from inactivated bacteria, such as an outer membrane protein extract. The Mannheimia haemolytica antigen may be a recombinantly-produced antigen, such as a recombinant leukotoxin (rLKT).

The present invention provides for an immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen, wherein the bovine influenza D virus antigen is an inactivated virus. The bovine influenza D virus antigen may be a modified, live virus. The modified, live bovine influenza D virus antigen may be a modified, live virus modified by codon deoptimization. A modified, live virus modified by codon deoptimization may have one or more deoptimized genomic segments with a cDNA sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

The present invention provides for an immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen, which further comprises at least one antigen from an additional bovine pathogen. The additional bovine pathogen may be a bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof.

The present invention provides for a vaccine for bovine respiratory disease comprising a bovine influenza D virus and a Mannheimia haemolytica antigen, and a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant. The Mannheimia haemolytica antigen may be an inactivated bacterium, or extracts from inactivated bacteria, such as an outer membrane protein extract. The Mannheimia haemolytica antigen may be a recombinantly-produced antigen, such as a recombinant leukotoxin. The bovine influenza D virus antigen may be an inactivated virus. The bovine influenza D virus antigen may be a modified, live virus. The modified, live bovine influenza D virus antigen may be a modified, live virus modified by codon deoptimization.

The present invention provides for a vaccine for bovine respiratory disease comprising a bovine influenza D virus and a Mannheimia haemolytica antigen, and a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant, which further comprises at least one antigen from an additional bovine pathogen. The additional bovine pathogen may be a bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof.

The present invention provides for a method of treating bovine respiratory disease, comprising administering to a bovine an immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen. The Mannheimia haemolytica antigen may be an inactivated bacterium, or extracts from inactivated bacteria, such as an outer membrane protein extract. The Mannheimia haemolytica antigen may be a recombinantly-produced antigen, such as a recombinant leukotoxin. The administering may be done orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly.

The present invention provides for a method of treating bovine respiratory disease, comprising administering to a bovine an immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen, wherein the bovine influenza D virus antigen is an inactivated virus. The bovine influenza D virus antigen may be a modified, live virus. The modified, live bovine influenza D virus antigen may be a modified, live virus modified by codon deoptimization. The administering may be done orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly.

The present invention provides for a method of treating bovine respiratory disease, comprising administering to a bovine an immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen, which further comprises at least one antigen from an additional bovine pathogen. The additional bovine pathogen may be a bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof. The administering may be done orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly.

The present invention provides for a method of treating bovine respiratory disease, comprising administering to a bovine a vaccine for bovine respiratory disease comprising a bovine influenza D virus and a Mannheimia haemolytica antigen, and a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant. The Mannheimia haemolytica antigen may be an inactivated bacterium, or extracts from inactivated bacteria, such as an outer membrane protein extract. The Mannheimia haemolytica antigen may be a recombinantly-produced antigen, such as a recombinant leukotoxin. The bovine influenza D virus antigen may be an inactivated virus. The bovine influenza D virus antigen may be a modified, live virus. The modified, live bovine influenza D virus antigen may be a modified, live virus modified by codon deoptimization. The administering may be done orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly.

The present invention provides for a method of treating bovine respiratory disease, comprising administering to a bovine a vaccine for bovine respiratory disease comprising a bovine influenza D virus and a Mannheimia haemolytica antigen, and a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant, which further comprises at least one antigen from an additional bovine pathogen. The additional bovine pathogen may be a bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof. The administering may be done orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly.

The present invention provides for the use of a combination of a bovine influenza D virus antigen and a Mannheimia haemolytica antigen in treating bovine respiratory disease. The Mannheimia haemolytica antigen may be an inactivated bacterium, or extracts from inactivated bacteria, such as an outer membrane protein extract. The Mannheimia haemolytica antigen may be a recombinantly-produced antigen, such as a recombinant leukotoxin. The bovine influenza D virus antigen may be an inactivated virus. The bovine influenza D virus antigen may be a modified, live virus. The modified, live bovine influenza D virus antigen may be a modified, live virus modified by codon deoptimization. The treatment may be done orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly. The treatment may be done therapeutically or prophylactically. The combination may further comprise a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant. The combination may further comprise at least one antigen from an additional bovine pathogen. The additional bovine pathogen may be a bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof.

The present invention provides for the use of a bovine influenza D virus antigen and a Mannheimia haemolytica antigen in the manufacture of a medicament for bovine respiratory disease. The Mannheimia haemolytica antigen may be an inactivated bacterium, or extracts from inactivated bacteria, such as an outer membrane protein extract. The Mannheimia haemolytica antigen may be a recombinantly-produced antigen, such as a recombinant leukotoxin. The bovine influenza D virus antigen may be an inactivated virus. The bovine influenza D virus antigen may be a modified, live virus. The modified, live bovine influenza D virus antigen may be a modified, live virus modified by codon deoptimization. The medicament may be administered orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly. The administration may be done therapeutically or prophylactically. The medicament may further comprise a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant. The medicament may further comprise at least one antigen from an additional bovine pathogen. The additional bovine pathogen may be a bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof.

The present invention provides for a composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen for use in the treatment, reduction or prevention of bovine respiratory disease in a bovine. The Mannheimia haemolytica antigen may be an inactivated bacterium, or extracts from inactivated bacteria, such as an outer membrane protein extract. The Mannheimia haemolytica antigen may be a recombinantly-produced antigen, such as a recombinant leukotoxin. The bovine influenza D virus antigen may be an inactivated virus. The bovine influenza D virus antigen may be a modified, live virus. The modified, live bovine influenza D virus antigen may be a modified, live virus modified by codon deoptimization. The composition may be administered orally, intranasally, intratracheally, or by injection, such as subcutaneously or intramuscularly. The composition may further comprise a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant. The composition may further comprise at least one antigen from an additional bovine pathogen. The additional bovine pathogen may be a bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof.

The term “bovine” when used to refer to an animal to which the invention is administered means any member of the genus Bos, including domestic cattle (Bos taurus).

As used herein “bovine influenza D virus” (IDV) means an RNA virus of the family orthomyxoviridae. IDV is considered to be a separate genus from influenzavirus A, B and C, although IDV appears to be closest related to influenzavirus C. The genome of an IDV contains seven linear negative-sense single-stranded RNA molecules. Two representative isolates of IDV are described in Table 1, without limitation of the invention claimed. As an RNA virus, IDV would be expected to have a high mutation rate leading to a process called “antigenic drift,” small changes in the genome that produce viruses with differing antigenic properties.

As used herein, the term “virus” could mean either the species of virus, or, interchangeably, an individual infectious unit, which may contain nucleic acids, proteins, and a lipid membrane. An individual infectious unit is also called a “viral particle” or a “virion”, the latter terms being synonymous.

As used herein, a “strain” or “isolate” a virus means a collection of genetically homologous virions. Two viruses would be considered “homologous” if those viruses map to the same phylogenetic clade. Two viruses would be considered “heterologous” if those viruses map to different phylogenetic clades. The degree of polymorphism among IDV isolates has not yet been evaluated.

TABLE 1 Representative isolates of IDV. % identity between Segment: NEB isolate KS isolate isolates PB2 2356 nt¹ 2337 nt 99% SEQ ID NO: 1 SEQ ID NO: 8 PB1 2261 nt 2307 nt 97% SEQ ID NO: 2 SEQ ID NO: 9 P3 2194 nt 2196 nt 99% SEQ ID NO: 3 SEQ ID NO: 10 HE 2049 nt 2045 nt 96% SEQ ID NO: 4 SEQ ID NO: 11 NP 1776 nt 1774 nt 99% SEQ ID NO: 5 SEQ ID NO: 12 P42 (matrix) 1219 nt 1218 nt 97% SEQ ID NO: 6 SEQ ID NO: 13 NS1 & NS2 869 nt 869 nt 99% SEQ ID NO: 7 SEQ ID NO: 14 ¹nt = nucleotides.

“Percent identity” can be determined by calculating the number of identical nucleotides at the same positions in a nucleic acid. Calculation of percent identity includes determination of the optimal alignment between two or more sequences. Computer programs and algorithms such as the Basic Local Alignment Search Tool (BLAST) may be used to determine the percent identity. BLAST one of the many resources provided by the U.S. National Center for Biotechnology Information. Because the genetic code is degenerate, and more than one codon can encode a given amino acid, coding regions of nucleic acids are considered identical if the nucleic acids encode identical polypeptides.

As used herein, the term “modified, live virus” applies to any individual viral particle (i.e. a “virion”) or to a multiplicity of viral particles whose genetic sequence has been altered from the genetic sequence of a naturally-occurring wild type virus. Alterations include, without limitation, genetic mutations such as insertions and deletions of nucleotides and transitions and transversions which change one nucleotide for another nucleotide. Alterations can be accomplished by adapting a wild-type virus to replication in a tissue culture system, and continuing to passage a virus in a tissue culture system, whereby the virus accumulates genetic mutations. Alterations can also be accomplished using molecular techniques. One such molecular technique involves artificially altering the codon usage of a virus, or “codon deoptimization.” Such techniques have been described, for example, in U.S. Pat. Nos. 8,846,051; 9,476,032; and US2016/024477 (U.S. Ser. No. 14/777,204), all of which are hereby incorporated by reference in their entirety.

Attenuated viruses form a subset of modified, live viruses. As used herein, the term “attenuated” or “attenuation” means the ability of virus to cause or exacerbate clinical disease has been reduced or eliminated. An attenuated virus can still infect a host cell, either in vitro or in vivo, and that infection may result in subclinical effects in the host organism, but that infection does not result in one or more clinical disease symptoms.

In contrast, as used herein, “inactivated” viruses or bacteria mean pathogens which can no longer replicate in a host. Inactivated pathogens are considered to be killed or dead. Inactivation can be accomplished by a variety of methods, including but not limited to chemical alteration, to chemical or physical alterations in the structure of a pathogen, or to chemical or physical alterations in the pathogen's genomic nucleic acids.

An “antigen” is any molecule capable of being specifically detected by the immune system of an organism. Typically, an antigen is a protein encoded by a pathogen's genome or derived from products of the pathogen's genome. An antigen may be an entire virion or bacterium, or a fraction thereof, or an isolated polypeptide. Antigens may be produced naturally or recombinantly. The presence of antigens can be specifically detected by the surface antigen receptors of both host T lymphocytes and host B lymphocytes and by antibody molecules synthesized by host cells. A non-limiting example of an IDV antigen would be the hemagglutinin-esterase glycoprotein. Non-limiting examples of Mannheimia antigens would be an extract of outer membrane proteins (i.e. a mixture of partially purified antigens) and a recombinantly expression leukotoxin polypeptide.

“Immunogenicity” refers to the ability of an antigen to elicit an immune response, said immune response comprising both antigen-specific responses and non-antigen-specific responses or innate immune responses. “Protective immunity” is an immune response which can reduce or prevent clinical symptoms when an immunized animal is challenged or exposed to a pathogenic strain. As one skilled in the art would appreciate, protective immunity may decline with time or increased age of the immunized animal. Protective immunity as used herein should be effective for at least four months, but preferably at least six months, from the latest date of immunization. Protective immunity may be elicited with a single dose of a vaccine. A second or further dose may be used to increase or prolong the protective immune response. For example, increasing the protective immune response in a breeding cow may result in an increased level of maternally derived antibody in calves.

In contrast to an antigen, an “adjuvant” is a non-specific stimulator of an immune response. An adjuvant could stimulate the innate immune response by binding and activating a pattern recognition receptor (PRR). Such stimulators of PRRs could be, for example, viral or bacterial nucleic acids, lipids from bacteria or parasites, or bacterial proteins or toxins, or any artificially-constructed mimic of such molecules. Adjuvants also include, without limitation: inorganic compounds that aggregate antigens to facilitate recognition by B lymphocytes or uptake by phagocytes, such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide or ammonium sulfate; oils; and detergents. Adjuvants could also be host mediators of immune signaling, such as, without limitation, cytokines, lymphokines, chemokines, interferons, anaphylatoxins, growth factors, differentiation factors, and adhesion molecules.

As used herein, an “immunogenic composition” is a composition that elicits an immune response when administered to an animal. An immunogenic composition comprises at least one antigen and at least one pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent. A description of pharmaceutically-acceptable excipients, stabilizers, solubilizers, or diluents can be found, for example, in “Remington: The Science and Practice of Pharmacy,” Lloyd V. Allen, ed., Pharmaceutical Press, London, UK, 22^(nd) edition, 2012. The antigen can be a whole virus, bacterium, or other pathogen, either live or inactivated. The antigen can also be isolated, purified, or a partially purified antigenic molecule from a virus, bacterium, or other pathogen. The antigen can be a polypeptide, a polysaccharide, a nucleic acid, or a lipid. The antigen is at least partially isolated from its natural environment, which could include factors capable of diluting, misdirecting or interfering with an effective immune response to the antigen.

As used herein, a “vaccine” is an immunogenic composition which stimulates an animal's immune system to develop protection from, resistance to, prevention of, or treatment for a disease symptom when administered to an animal in need thereof, wherein said symptom is caused by a pathogenic organism, for example a virus or a bacterium. A vaccine may include, without limitation, viral antigens or intact virions, either live or inactivated, in composition with pharmaceutically-acceptable adjuvants, excipients, stabilizers, solubilizers, or diluents, and optionally an adjuvant. A vaccine may include, without limitation, bacterial antigens or intact bacteria, either live or inactivated, in composition with pharmaceutically-acceptable adjuvants, excipients, stabilizers, solubilizers, or diluents, and optionally an adjuvant. A vaccine may include multiple antigens from multiple pathogens.

As used herein, the terms “treating”, “to treat”, or “treatment”, include restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. A treatment may be applied prophylactically or therapeutically.

As used herein, “administering” includes but is not limited to cutaneous, subcutaneous, intramuscular, mucosal, submucosal, transdermal, intratracheal, oral or intranasal administration. Administration could occur by injection or by topical administration.

The following experimental examples are illustrative of bovine respiratory disease vaccines. It will be appreciated that other embodiments and uses will be apparent to those skilled in the art and that the invention is not limited to these specific illustrative examples or preferred embodiments.

EXAMPLE 1

This study evaluated if respiratory pathogenesis develops in 6-9 month old cattle when administered a high titer influenza D virus (IDV) and if that pathogenesis can be mitigated by a homologous inactivated vaccine.

A total of 83 Holstein or Holstein cross cattle approximately 6 months old were used for this study. The animals were allowed to acclimate for a minimum of 5 days to their new environment at the testing facility. Housing consisted of an ABSL-2 containment room, with common feed line bunk and open water tank system in each room. The animals met the following inclusion criteria: BVDV-PI negative via ear notch and antigen capture ELISA (ACE); in good health based on physical examination conducted on Day 0; and seronegative to IDV. Animals were randomly allocated to treatment groups (Table 2) using a random allocation plan. Study personnel conducting clinical observations, collecting samples and analyzing samples were masked to treatment identity. Following administration of each treatment (e.g. vaccination), animals were housed by treatment group such that each group was in an isolated pen with no physical contact between each treatment group, with the exception of calves from treatment group four (T04), which were co-housed with challenged cattle (groups T02 and T03).

TABLE 2 Experimental design. Serial slaughter Groups Treat- Vaccina- Chal- Serol- (post-challenge days) ment tion lenge ogy 37 d 39 d 41 d 43 d 45 d groups day 35 d 14-96 d (2) (4) (6) (8) (10) T01 0, 14 No 10 head 3 hd 3 hd 3 hd 3 hd 3 hd (hd) T02 No IDV — 5 hd 5 hd 5 hd 5 hd 5 hd T03 0, 14 IDV — 5 hd 5 hd 5 hd 5 hd 5 hd T04 No No — — — — 3 hd 4 hd

The experimental vaccine comprised beta-propriolactone (BPL) inactivated IDV strain D/Bovine/KS/14-22/2012. Vaccine was standardized to 640 hemagglutinatin units (HAU) per dose. Antigen was batched and formulated/adjuvanted with 30% EMULSIGEN® D. The vaccine underwent a modified sterility test consisting of final product being plated on blood agar, incubated at 37° C. and examination of colonies formed approximately 48 hours later. No colonies were identified and product considered sterile. Calves were vaccinated (2.0 mL) by subcutaneous (SQ) injection on the right side of the neck (20-gauge 1-inch needle) on Day 0 and Day 14. Animals were observed at approximately one (1) hour post-vaccination to assess response to vaccination. No abnormal events were noted.

Challenge occurred on Day 35 (˜7 months of age). On Day 35 (35 d) each calf was challenged intranasally (5.0 ml; 2.5 mL/nostril) with IDV strain D/Bovine/KS/14-22/2012 at 10⁶ TCID₅₀.

All animals were evaluated for depression, body condition and respiratory distress on Days 35 through necropsy and scored. Animals were evaluated and scores recorded prior to movement or feeding. Rectal body temperatures were recorded daily during the challenge period (Day 35-45). Temperatures were recorded at approximately the same time each day. One blood sample was collected at least 7 days prior to arrival at the facility and the rest of the blood samples for the determination of IDV hemagglutination inhibition were collected on Days 0, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 98, 112. One nasal swab sample was collected at least 7 days prior to arrival at the facility and the rest of the nasal swabs were collected from all animals on Days 35 (pre-challenge), 37, 39, 41, 43, and 45. Swabs were placed into a tube containing transport medium. Tubes were labeled with date of collection, animal identification (ear tag number), sample type (nasal), and study number. Tubes were cooled immediately by placing them into a cooler containing ice packs. The tubes were stored at −70° C.

On Days 35, 37, 39, 41, 43, and 45, the respective animals were humanely euthanized for lung lesion scoring, collection of bronchoalveolar lavage (BAL) fluid, and the respective tissue. Lungs were removed from each animal. BAL fluid was collected aseptically from the excised lungs. Sterile phosphate buffered saline (PBS) was pipetted into the lungs and pipetted back as the lungs are gently massaged. The BAL fluid extracted was placed into a tube kept on ice. Each sample of BAL fluid was divided into two aliquots and labeled with animal ID and date of sample collection. Both set of aliquots were stored frozen (≤−20° C.).

Each of the seven pulmonary lobes was examined both visually and by palpation for gross characteristic lesions attributable to IDV. The amount of lesion/consolidation in each pulmonary lobe was scored and recorded as an actual value between 0 and 100% of the lobe. The amount of lesion (score) in each lobe was inserted in a weighted formula in order to calculate the percentage of lung with lesions. The weight assigned to each of the seven lobes (according to the relative weight of the lung lobes) is as follows: left apical lobe=10%; left cardiac lobe=10%; left diaphragmatic lobe=25%; right apical lobe=10%; right cardiac lobe=10%; right diaphragmatic lobe=25%; and accessory lobe=10%. The addition of the weighted percentages resulted in a composite lung lesions score.

Lung, turbinate, and tracheal tissues were examined at necropsy for lesions and a section was excised for immunohistochemistry. The tracheobronchial lymph node was excised for immunohistochemistry evaluation as well. All samples were stained with hematoxylin and eosin for structural evaluation as well as stained for IDV virus using standard immunohistochemistry techniques.

Nasal swabs were evaluated for IDV virus via RT-PCR using a specific primer set developed by the Kansas State University (KSU) veterinary diagnostic laboratory. The mean with standard deviation of the threshold cycle (CT) value of the tested nasal swabs were extrapolated using a standard curve of known tissue culture infectious dose (TCID₅₀) IDV virus to produce a viral titer value of the nasal swabs. The CT value was extrapolated using a standard curve of known tissue culture infectious dose (TCID₅₀) IDV virus to produce a viral titer value (Table 3).

Bronchioalveolar lavage fluid (Table 4) and tracheal swabs (from days 41, 43, and 45) (Table 5) were also evaluated for IDV virus via RT-PCR, and the CT value was extrapolated using a standard curve of known tissue culture infectious dose (TCID₅₀) IDV virus to produce a viral titer value for each sample group.

TABLE 3 Nasal Swab Viral Titer (TCID₅₀). At 2 4 6 8 10 Treatment Group challenge DPC¹ DPC DPC DPC DPC T01- Vaccination+ 0 0 0 0 0 0 Challenge− T02- Vaccination− 0 2.64 5.68 3.765 0.24 0.275 Challenge+ T03- Vaccination+ 0 1.52 3.98 2.01 1.23 0 Challenge+ T04- Contact 0 0 0.65 0.78 3.34 4.95 Controls ¹Days post challenge (DPC).

TABLE 4 Tracheal Swab Viral Titer (TCID₅₀). Treatment Group 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0 0 0 Challenge− T02- Vaccination− 5.34 0.64 0.18 Challenge+ T03- Vaccination+ 1.72 0.64 0 Challenge+ T04- Contact NA 4.63 3.3 Controls

Assessments of the upper respiratory tract and lung lesion scoring as outlined above were taken by the blinded study investigator. Composite lung lesions scores are outlined in Table 6 as mean composite scores/number of animals per group at each time point.

TABLE 5 BALF Viral Titer (TCID₅₀). Treatment Group 2 DPC 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0 0 0 0 0 Challenge− T02- Vaccination− 0.54 2.68 4.98 1.52 1.26 Challenge+ T03- Vaccination+ 0 1.22 0 0 0 Challenge+ T04- Contact NA NA NA 1.73 3.15 Controls

TABLE 6 Mean Composite Lung Lesion Scores Treatment Group 2 DPC 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0.59/3 0.00/3 0.24/3 0.06/3 0.04/3 Challenge− T02- Vaccination− 0.29/5 1.01/5 2.46/5 0.64/5 1.70/5 Challenge+ T03- Vaccination+ 0.18/5 0.72/5 1.34/5 0.58/5 1.04/5 Challenge+ T04- Contact NA NA NA 0.59/3 0.15/2 Controls

Mild lung lesion scores were observed with slight trending differences between the vaccinates versus challenged animals. Stronger statistical power could be added as well as refinement of the challenge model to further explore the gross lung pathology caused by IDV. Gross pathological examinations of the upper respiratory tract, exclusively the trachea demonstrated a mucoid exudate with peak observations at 4 and 6 days post challenge.

Fixed tissue samples from the nasal turbinate and the trachea were processed, hematoxylin and eosin stained, and then stained with antibody specific for IDV virus. The number of positive samples out of total number of animals per group for nasal turbinate samples and for trachea samples are represented in Tables 7 and 8, respectively.

TABLE 7 Immunohistochemistry Results (number positive) of Nasal Turbinate Samples 2 Days Post Challenge Treatment Group (DPC) 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0/3 0/3 0/3 0/3 0/3 Challenge− T02- Vaccination− 1/5 1/5 5/5 1/5 0/5 Challenge+ T03- Vaccination+ 0/5 0/5 0/5 0/5 0/5 Challenge+ T04- Contact NA NA NA 1/3 2/2 Controls

TABLE 8 Immunohistochemistry Results (number positive) of Trachea Samples 2 Days Post Challenge Treatment Group (DPC) 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0/3 0/3 0/3 0/3 0/3 Challenge− T02- Vaccination− 0/5 4/5 5/5 0/5 0/5 Challenge+ T03- Vaccination+ 0/5 2/5 0/5 0/5 0/5 Challenge+ T04- Contact NA NA NA 1/3 0/2 Controls

Serological antibody titers were assessed at study day (SD) 0, 14, 21, 28, 35, and 42 for serological conversion to the vaccine. Geometric mean titer (GMT) of hemagglutination inhibition (HI) titer of the serum from each group is depicted in Table 9. Serological evaluation shows there was an increase in HI titer against IDV in the vaccinated groups compared to the non-vaccinates. Routine dose and vaccine optimization could be evaluated further to possibly increase the antibody titer. For comparison, in humans a HI titer of 40 (5.3 log₂) is generally considered protective against influenzavirus A infection but does not fully alleviate disease symptoms.

TABLE 9 Hemagglutination Inhibition (HI) Serum Titer (log₂). At 14 21 28 35 42 Treatment Group challenge DPC DPC DPC DPC DPC T01- Vaccination+ 28 58.7 154.7 210.7 160 160 Challenge− T02- Vaccination− 28.8 37.6 24 32 34.4 40 Challenge+ T03- Vaccination+ 28.8 49.6 132.8 201.6 208 328 Challenge+ T04- Contact 32 48 32 36 40 36 Controls

Clinical observations of depression and body condition scoring were mostly unremarkable. Respiratory distress was demonstrated in the challenged animals with peak clinical signs at 6 days post challenge. No differences in respiratory distress between vaccinated and unvaccinated animals were observed.

The primary objective of this study was to assess if bovine influenza D virus (IDV) was capable of eliciting clinical pathogenesis and discover if a homologous inactivated vaccine preparation was able to prevent the clinical pathogenesis. In animals challenged with IDV, replication of the virus took place in the upper respiratory tract based on RT-PCR. The kinetics of viral replication indicate that the highest viral titers were shown at 6 days post challenge. The amount of virus was trending towards being higher in the nasal swabs, tracheal swabs, and the BALF of treatment group 2 (T02) as compared to the vaccinated groups with the greatest differences in the tracheal swabs and BALF samples. Additionally, immunohistochemistry staining of IDV was performed on the fixed excised tissues of the upper respiratory tract (turbinate and trachea). Challenged animals were positive for IDV virus via staining with five of five cattle being positive at 6 days post challenge. No animals receiving vaccine were positive for IDV via IHC staining. It can be hypothesized that immunization may limit the amount of viral spread by reducing the viral load in the upper respiratory tract. Further, the contact control group (T04) showed that the virus was passed from the challenged animals (T02) to co-housed naïve animals.

Based on the results of this study IDV appears to replicate in the upper respiratory tract and elicit a mild inflammation based on immunohistochemical observations. This viral infection may be an important contributor to the bovine respiratory disease complex in which preventive intervention like vaccines could increase overall cattle health.

EXAMPLE 2

This study evaluated the hypothesis that the upper respiratory infection caused by Influenza D virus is a precursor to, or exacerbator of, a more severe secondary Mannheimia haemolytica infection.

A total of 70 Holstein or Holstein cross cattle 5-9 months old were used for this study. The animals were allowed to acclimate for a minimum of 5 days to their new environment at the testing facility. Housing consisted of an ABSL-2 containment room, with common feed line bunk and open water tank system in each room. The animals met the following inclusion criteria: BVDV-PI negative via ear notch and antigen capture ELISA (ACE); in good health based on physical examination conducted on Day 0; seronegative to IDV; and seronegative to leukotoxin (LKT) of M. haemolytica.

The IDV experimental vaccine comprised BPL inactivated IDV strain D/Bovine/NE/103795/2012. The strain of influenza virus used for immunizations and challenge was changed from D/Bovine/KS/14-22/2012 to D/Bovine/NE/103795/2012 due to the latter virus growing better for scale up. Vaccines were standardized to 1280 HAU per dose. Antigen was batched and formulated/adjuvanted 1:1 weight by weight with SEPPIC MONTANIDE ISA 201 VG. The vaccine underwent a 7-day media bottle sterility test in Fluid Thioglycollate Media (FTM) and Tryptic Soy Broth (TSB) broth prior to use.

The M. haemolytica vaccine comprised an inactivated outer membrane proteins (OMP) extract and recombinant LKT (rLKT). Vaccines were standardized to 250 μg of OMP and 125 μg of LKT per dose. Antigen was batched and formulated/adjuvanted 1:1 weight by weight with SEPPIC MONTANIDE ISA 201 VG. The vaccine underwent a 7-day media bottle sterility test in FTM and TSB broths prior to use.

TABLE 10 Treatment groups. IDV M. haemolytica Necropsy and Treatment Vaccine Animals/ Dose/ Challenge Challenge Lung Scores Group (SD 0 & 14) Treatment Route (SD 35) (SD 41) (SD 48) T01 Adjuvanted 10 2 mL/SC + +* + Saline T02 Saline 10 2 mL/SC + −  + T03 Saline 9 2 mL/SC − +* + T04 Adjuvanted 10 2 mL/SC −  +** + Saline T05 IDV vaccine 10 2 mL/SC + +* + T06 M. haemolytica 10 2 mL/SC + +* + vaccine T07 M. haemolytica + 10 2 mL/SC + +* + IDV vaccine *Mannheimia haemolytica challenge administered intranasally with disposable atomizer (5.0 mL; 2.5 mL/nostril) and 5.0 mL intratracheally with endoscope to be delivered at the larynx/trachea **Mannheimia haemolytica challenge administered intratracheally by locating the bifurcation of the trachea and pulling back approximately 10 cm and delivering 60 mL with M. haemolytica strain

The combination vaccine comprised BPL inactivated IDV strain D/Bovine/NE/103795/2012 plus inactivated outer membrane proteins (OMP) extract and rLKT. Vaccines were standardized to 1280 HAU per dose of IDV, 250 μs of OMP, and 125 of LKT per dose. Antigens were batched and formulated/adjuvanted 1:1 weight by weight with SEPPIC MONTANIDE ISA 201 VG. The vaccine underwent a 7-day media bottle sterility test in FTM and TSB broth prior to use.

All candidate animals that met selection criteria and inclusion/exclusion criteria were eligible for enrollment. Animals were randomly allocated to treatment groups using a random allocation plan. Study personnel conducting clinical observations, collecting samples and analyzing samples were masked to treatment identity. The study investigator who performed the lung lesion scoring was blinded to the specific calf ID during scoring.

Following arrival at the study site, animals were housed within three ABSL-2 containment rooms. Groups T01, T02, T05, T06 and T07 were housed in two rooms (25 animals per room) with equal representation of treatment groups between rooms. Groups T03 and T04 were housed in a separate third room to prevent shedding of IDV from challenged animals to cross contaminate IDV naïve animals. Animals were comingled within the three rooms. Following M. haemolytica challenge on study day 41 (SD41) the entire herd was resorted to equally distribute treatment groups (T01-T07) between the three ABSL-2 containment rooms with 23-24 animals per room. Equal representation of each treatment group (3-4 animals per group per room) was obtained following the resort. Calves were vaccinated (2.0 mL) by subcutaneous (SQ) injection on the right side of the neck (18-gauge 1-inch needle) on Day 0 and left side of the neck on Day 14. Animals were observed at approximately one (1) hour post-vaccination to assess response to vaccination. Study animals were observed daily to determine general health status beginning on Day −5 and continuing through Day 48. Observations were made at approximately the same time each day. Animal health observations consisted of pen-side visual assessments of animals for indicators of animal health.

Influenza D Virus challenge occurred on Day 35 (˜6-10 months of age) for Groups T01, T02, T05, T06, and T07. On Day 35, each calf was challenged intranasally with disposable PREVAL® can atomizer (10.0 mL; 5 mL/nostril) with IDV strain D/Bovine/NE/103795/2012 at 10^(5.7) TCID₅₀.

IDV was produced as follows. Three 850 cm² tissue culture polystyrene roller bottles were planted with ST-C cells at a target density of 2×10⁷ cells/bottle (i.e. 23529 cells/cm²) in EMEM media with 7.5% FBS and 4 mM L-glutamine. The roller bottles rotated at 0.25 rpm at 37° C. After 7 days, the cells were infected. To infect, the used media was first replaced with pre-warmed EMEM media only, and IDV (D/Bovine/NE/103795/2012) was added at a multiplicity of infection (MOI) of 0.05. After an additional 3 days of incubation at 0.25 rpm and 37° C., the entire roller bottle and contents were harvested by freezing at −80° C., thawing and dispensed aseptically.

At time of harvest, samples were taken for virus titer assay. To assay, ST-C cells were planted at 15,000 cells/well in 96-well plates. 3 days post-planting, when the cells were ˜95% confluent, the wells were washed with PBS two times before 100 μL EMEM with 0.2 mL/L gentamicin was added to each well. Next, the harvested IDV samples were serially diluted 1:10 before 100 μL of each dilution was added to each well. After an additional 3 days of incubation at 37° C. and 5% CO₂, the plates were fixed with 80% acetone at −20° C. for at least 20 minutes, before being dried at 37° C. For staining, the primary antibody was diluted in D-PBS, added to each well, and incubated at 37° C. for 1 hour. Following three washes in PBS, the secondary antibody was diluted in D-PBS, added to each well, and incubated at 37° C. for 75 minutes. After two washes in PBS, the results were obtained visually under a fluorescent microscope and calculated via the Spearman-Karber equation.

Mannheimia haemolytica Challenge occurred on Day 41 (˜6-10 months of age) for Groups T01, T03, T04, T05, T06, and T07. On Day 41, each calf from Groups T01, T03, T05, T06, and T07 was challenged intranasally with a disposable atomizer (5.0 mL; 2.5 mL/nostril) and 5.0 mL intratracheally with an endoscope at the larynx/trachea bifurcation with M. haemolytica strain at 10⁹ CFU/mL. On Day 41, each calf from Group T04 was challenged intratracheally by locating the bifurcation of the trachea and pulling back approximately 10 cm and delivering 60 mL of M. haemolytica strain at 10⁹ CFU/mL with endoscope.

Rectal body temperatures were recorded daily during the challenge period (Day 35-48). Temperatures were recorded at approximately the same time each day.

One blood sample was collected at a minimum of 7 days prior to arrival at the facility and the rest of the blood samples for the determination of IDV hemagglutination inhibition and anti-M. haemolytica LKT antibody were collected on Days −2, 14, 21, 28, 35, 41, and 48.

One nasal swab sample was collected at a minimum of 7 days prior to arrival at the facility and the rest of the nasal swabs were collected from all animals on Days 35 (pre-challenge), 37, 39, 41, 43, 45 and 47. In addition, swabs of the trachea were obtained at necropsy on Day 48.

On Day 48, the indicated animals were humanely euthanized for lung lesion scoring, collection of bronchoalveolar lavage (BAL) fluid, and tissue collection. Lungs were removed from each animal, and BAL fluid was collected aseptically from the excised lungs. Sterile phosphate buffered saline (PBS) was pipetted into the lungs and collected by pipette as the lungs were gently massaged. The BAL fluid extract was placed into a tube kept on ice. Each sample of BAL fluid was divided into two aliquots and labeled with animal ID and date of sample collection. Both set of aliquots were stored frozen (≤−20° C.).

Each of the seven pulmonary lobes was examined both visually and by palpation for gross characteristic lesions attributable to M. haemolytica and/or IDV. The amount of lesion/consolidation in each pulmonary lobe was scored and recorded as an actual value between 0 and 100% of the lobe. The amount of lesion (score) in each lobe was inserted in a weighted formula in order to calculate the percentage of lung with lesions. The weight assigned to each of the seven lobes (according to the relative weight of the lung lobes) is as follows: left apical lobe=10%; left cardiac lobe=10%; left diaphragmatic lobe=25%; right apical lobe=10%; right cardiac lobe=10%; right diaphragmatic lobe=25%; and accessory lobe=10%.

Nasal turbinates, trachea, tracheobronchial lymph node, and lung tissues were examined at necropsy for lesions and a section of each was excised. The section was divided so that one half was placed in fixative (buffered formalin or equivalent) for immunohistochemistry evaluation as well as stained with hematoxylin and eosin for structural evaluation, and the other half will be placed in RNALATER® or frozen at −70° C. for cytokine and inflammatory gene PCR analysis.

The primary variable, total percent lung lesion score on SD48, was calculated and analyzed as in Example 1. Secondary variables were summarized or analyzed including daily depression scores, daily body condition score, daily respiratory score and daily rectal body temperature from SD 35 to SD48 was well as mortality and removals from SD 35 to SD48.

Data from the seven treatments T01, T02, T03, T04, T05, T06 and T07 were pooled and analyzed. For each variable, calculations were done on a per animal basis because the animal was the experimental unit. All testing was conducted at the 5% significance level unless otherwise stated.

Total percent lung lesion scores on Day 48 were transformed using arsin (sqrt(Total Percent Lung Score)) and were analyzed using a linear mixed model analysis (SAS® PROC MIXED v 9.4 or higher), with fixed effect, per treatment. Linear contrasts were conducted using the following pairwise comparisons:

T01 vs. T02, T03, T04;

T02 vs. T03, T04;

T03 vs. T04; and

T04 vs. T05, T06, and T07.

Least squares means (LSMEANs) and standard errors as well as arithmetic means and standard deviations were calculated for each treatment group and back-transformed results are presented.

In addition, summary statistics were presented for the mitigated fractions (MF) (R version 3.0.3 (2014 Mar. 6)). Frequency distributions of daily depression scores, daily body condition score and daily respiratory scores were obtained for each time by treatment.

There were two mortalities due to morbidity, both in Treatment Group 4. No other groups had mortalities.

The primary objective of this study was to evaluate the mortality and lung lesion scores of the different treatment groups and to evaluate the hypothesis that the upper respiratory infection caused by Influenza D virus is a precursor to a more severe secondary Mannheimia haemolytica infection. Mean lung lesion scores of animals challenged intranasally with M. haemolytica following an Influenza D virus challenge showed a greater than fourfold increase compared to animals intranasally challenged with M. haemolytica alone (Group T01=5.6 and Group T03=1.2 respectively). This supports the hypothesis that an upper respiratory infection with an IDV leads to an increased potential for M. haemolytica to migrate from the upper respiratory tract where it typically colonizes to the lower respiratory tract and lungs where pathology starts to be exhibited.

The mean lung lesion score in the IDV and M. haemolytica challenged cattle was lower than what is observed in the traditional model of M. haemolytica infection in which a larger volume of bacteria (60 mL compared to 10 mL intranasal/intratracheal) is delivered directly to the lungs. The differences in the severity can be explained by the decreased dose and the anatomical and mechanical immunological defenses between the upper respiratory tract and the lungs.

TABLE 11 Mean Lung Lesion Scores IDV M. haemolytica Mean Treatment Challenge Challenge Lung Group Vaccine (SD 0 & 14) (SD 35) (SD 41) Scores T01 Adjuvanted Saline + +* 5.6 T02 Saline + −  0.5 T03 Saline − +* 1.2 T04 Adjuvanted Saline −  +** 23.9 T05 IDV vaccine + +* 2.5 T06 M. haemolytica + +* 0.9 vaccine T07 M. haemolytica + + +* 0.1 IDV vaccine *Mannheimia haemolytica challenge administered intranasally with disposable atomizer (5.0 mL; 2.5 mL/nostril) and 5.0 mL intratracheally with endoscope was delivered at the larynx/trachea **Mannheimia haemolytica challenge administered intratracheally by locating the bifurcation of the trachea and pulling back approximately 10 cm and delivering 60 mL with M. haemolytica strain

TABLE 12 Mitigated Fraction Statistical Analysis of Lung Lesion Scores Group versus Group MF Lower Bound Upper Bound T04 T05 0.90 0.66 1.00 T04 T06 0.92 0.70 1.00 T04 T07 1.00 1.00 1.00 T01 T05 0.42 −0.06 0.83 T01 T06 0.44 −0.06 0.86 T01 T07 0.70 0.28 1.00

Additionally, a combination inactivated vaccine containing both IDV and M. haemolytica antigens provided statistically significant protection (as measured by MF) to the dual challenge of lung lesion scoring that would be considered efficacious as the current statistical criteria outlines for M. haemolytica models used to license vaccines (>0.4 MF with positive upper and lower bounds). Monovalent vaccines of IDV (mean lung lesion score of 2.5) and M. haemolytica (mean lung lesion score of 0.9) provided a MF above the criteria but the upper and lower confidence bounds did not meet the satisfactory criteria. Therefore, a combination vaccine of IDV and M. haemolytica is an effective approach to controlling bovine respiratory disease. 

What is claimed is:
 1. An immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen.
 2. The immunogenic composition of claim 1, wherein the Mannheimia haemolytica antigen is selected from the group of an inactivated bacterium, an extract of outer membrane proteins, and a recombinant leukotoxin, and any combination thereof.
 3. The immunogenic composition of claim 1 or 2, wherein the bovine influenza D virus antigen is an inactivated virus.
 4. The immunogenic composition of claim 1 or 2, wherein the bovine influenza D virus antigen is a modified, live virus.
 5. The immunogenic composition of claim 4, wherein the modified, live bovine influenza D virus antigen is a modified, live virus modified by codon deoptimization.
 6. The immunogenic composition of claim 4, wherein the modified, live bovine influenza D virus antigen comprises at least one codon-deoptimized genomic segment having a cDNA sequence selected from the group of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.
 7. A vaccine for bovine respiratory disease comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen, and a pharmaceutically-acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant.
 8. The vaccine of claim 7, further comprising at least one antigen from an additional bovine pathogen.
 9. The vaccine of claim 8, wherein the additional bovine pathogen is selected from the group of bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PI3), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof.
 10. A method of treating bovine respiratory disease, comprising administering to a bovine the immunogenic composition or vaccine of any previous claim.
 11. The method of claim 10, wherein the administering is done orally, intranasally, intratracheally, or by injection.
 12. Use of a combination of a bovine influenza D virus antigen and a Mannheimia haemolytica antigen in treating bovine respiratory disease.
 13. Use of a bovine influenza D virus antigen and a Mannheimia haemolytica antigen in the manufacture of a medicament for bovine respiratory disease.
 14. A composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen for use in the treatment, reduction or prevention of bovine respiratory disease in a bovine.
 15. Use of the immunogenic composition of claims 1-6 or the vaccine of claims 7-9 in treating bovine respiratory disease.
 16. Use of the immunogenic composition of claims 1-6 or the vaccine of claims 7-9 in the manufacture of a medicament for bovine respiratory disease.
 17. A composition comprising the immunogenic composition of claims 1-6 or the vaccine of claims 7-9 for use in the treatment, reduction or prevention of bovine respiratory disease in a bovine. 